Tribrominated Diphenyl Ether (BDE-28) - American Chemical Society

Nov 5, 2013 - Metabolites of 2,4,4′-Tribrominated Diphenyl Ether (BDE-28) in. Pumpkin after In Vivo and In Vitro Exposure. Miao Yu, Jiyan Liu,* Than...
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Metabolites of 2,4,4′-Tribrominated Diphenyl Ether (BDE-28) in Pumpkin after In Vivo and In Vitro Exposure Miao Yu, Jiyan Liu,* Thanh Wang, Jianteng Sun, Runzeng Liu, and Guibin Jiang State Key Laboratory of Environmental Chemistry and Ecotoxicology, Research Center for Eco-Environmental Sciences, Chinese Academy of Sciences, Beijing, 100085, China S Supporting Information *

ABSTRACT: There is currently limited knowledge on PBDE metabolism in plants although they could play an important role in the environmental transformation of these persistent organic pollutants. In this study, pumpkin (Cucurbita maxima × C. moschata) was chosen as the model to understand the fate of BDE-28 in plants. MeO-tri-BDEs, OH-tri-BDEs, and OH-tri-BDEs were found as metabolites in plant samples of both in vivo hydroponic and in vitro tissue culture exposure. Three MeO-tri-BDEs were further identified as para-substituted metabolites. MeO-BDEs and OH-BDEs, respectively, accounted for about 1.6% and 1.5% (recovery corrected) of initial amount of BDE-28 according to the semiquantitative results. Other PBDEs, especially less brominated PBDEs as impurities in the standard of BDE-28, were also detected. The impurities and evaporation of the standard must be considered when trace metabolites are studied in exposure experiments.

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INTRODUCTION As a class of additive brominated flame retardants, polybrominated diphenyl ethers (PBDEs) have been widely used in many commercial products.1 The penta- and octa-BDE technical mixtures have recently been listed in the Stockholm Convention on Persistent Organic Pollutants (SC-POPs) in May 20092,3 due to their persistence, bioaccumulation and toxicity. As analogues of PBDEs, hydroxylated polybrominated diphenyl ethers (OH-PBDEs) and methoxylated polybrominated diphenyl ethers (MeO-PBDEs), have also been detected in various environmental matrices.4−10 However, the relationships between OH-PBDEs, MeO-PBDEs, and corresponding PBDEs are still a matter of discussion,11,12 which is important to elucidate the fate of PBDEs in the environment. Metabolic transformation of organic contaminant in plants is an important biotransformation process in ecosystems.13 However, only limited studies have focused on the metabolism of PBDEs in plants. Wang et al. found six debromination products, seven hydroxylated metabolites and four methoxylated products in maize after exposure to three BDE congeners.14 Our previous work12 investigated the metabolism of 2,2′,4,4′-tetrabromodiphenyl ether (BDE-47) in young whole pumpkin plant and found 2,4,4′-tribrominated diphenyl ether (BDE-28) as a debromination product, four hydroxylated metabolites and a methoxylated product. The fate of PBDEs in the plants affects the behavior of PBDEs in the food web because the plants are the primary trophic level. Thus studies on the metabolism of PBDEs in the plants not only provide the information on health and environmental risks of PBDEs but also supply an important clue for the remediation of those contaminants. © 2013 American Chemical Society

In vivo exposure of intact plants is an effective method to study the fate of certain compounds in plants, and can provide information on the distribution of parent compounds and metabolites. However, in vivo studies12,14−17 cannot completely eliminate the effects of microorganisms even with rigorous protocols. In vitro studies using tissue culture under axenic conditions can usually solve the problems caused by microbes. However, it is usually difficult to extrapolate the results from in vitro to in vivo conditions due to the complexity of the study organisms. Therefore, integrating in vivo and in vitro studies is an effective way to explain the metabolism of contaminants in plants. Some lower brominated diphenyl ethers such as 2,4,4′tribrominated diphenyl ether (BDE-28) are frequently detected in the environment at relatively high concentrations18,19 and tend to be bioaccumulative.20 BDE-28 was also found as a debromination product of BDE-47 in the pumpkin plant.12 Pumpkin has also been found to have a high bioaccumulation capacity for POPs such as polychlorinated biphenyls (PCBs)21 and DDT.22 Therefore, intact pumpkin (Cucurbita maxima × C. moschata) seedlings and tissue cultures were hydroponically exposed to BDE-28 to study the fate of BDE-28 in plants in this work.

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EXPERIMENTAL SECTION Chemicals. The standard of BDE-28 (solid form, 5 mg, 99.3%) and standard solutions of potential BDE metabolites Received: Revised: Accepted: Published: 13494

September 17, 2013 October 31, 2013 November 5, 2013 November 5, 2013 dx.doi.org/10.1021/es404144p | Environ. Sci. Technol. 2013, 47, 13494−13501

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after 10 days and sampled as 4 parts as shown in Figure 1. Root samples were carefully rinsed with deionized water. The rinsewater was combined with the exposure solutions for analysis of the remaining BDE-28 in the solution.

(BDE-1, BDE-2, BDE-3, BDE-4, BDE-5, BDE-6, BDE-7, BDE8, BDE-9, BDE-10, BDE-11, BDE-12, BDE-13, BDE-14, BDE15, BDE-16, BDE-17, BDE-18, BDE-19, BDE-20, BDE-21, BDE-22, BDE-23, BDE-24, BDE-26, BDE-27, BDE-29, BDE30, BDE-31, BDE-32, BDE-33, BDE-34, BDE-35, BDE-36, BDE-37, BDE-38, BDE-39), hydroxylated metabolites (2′-OHBDE-3, 2′-OH-BDE-7, 3′-OH-BDE-7, 2′-OH-BDE-28, 3′− OH-BDE-28), methoxylated metabolites (2′-MeO-BDE-3, 2′MeO-BDE-7, 3′-MeO-BDE-7, 4′-MeO-BDE-17, 2′-MeOBDE28, 3′-MeO-BDE-28), surrogate standards (BDE-25, 4′OH-BDE-17), and internal standards (5′-OH-BDE-25, 5′MeO-BDE-25) were all purchased from AccuStandard (New Haven, CT, USA). Their molecular structures are shown in Supporting Information Figure S1. The stock solution of BDE28 was prepared at 1250 μg/mL in methanol. The working solution for exposure was prepared by gradual dilution of the stock solution of BDE-28 by methanol to 100 μg/mL. For quantitative analysis, the working solutions of PBDEs and MeO-PBDEs were prepared at 1 μg/mL in hexane, and OHPBDEs were at 1 μg/mL in acetonitrile. Anhydrous sodium sulfate (Na2SO4) was heated for 6 h at 660 °C before use. Florisil (60−100 mesh, Sigma−Aldrich, St Louis, MO) was activated at 450 °C for 12 h and deactivated by adding 1% (w/w) water. All solvents used, including methyl tert-butyl ether (MTBE), dichloromethane (DCM), hexane, methanol, acetonitrile, acetone were of chromatographic grade and purchased from J.T. Baker (Phillipsburg, NJ) and Honeywell Burdick & Jackson (Seelze, Germany). 1Naphthalene acetic acid (NAA, 98%) and 6-Benzylaminopurine (6-BA, 99%), Phytagel and Murashige and Skoog medium (MS-medium) were bought from Sigma−Aldrich. Agarose were obtained from Biowest (Spain) and sucrose (ultrapure grade) was from Amresco LLC (Solon, OH). Ultrapure water (18.2 Ω) was produced by ultrapure water purification system (Barnstead International, Dubuque, IA). All other chemicals and reagents used were of analytical grade. In Vivo Hydroponic Exposure. Pumpkin (Cucurbita maxima × C. moschata) seeds, purchased from Yinong seed Co., LTD (Taiyuan, China), were ripened and rinsed with autoclaved deionized water (55 °C, 1 h) and then pregerminated on sterilized perlite beds (NaClO solution with 1% active Cl) for 7 days. The seedlings were used for exposure after growing to around 5 cm in height. Blank controls (with seedlings but without BDE-28) and unplanted controls (with BDE-28 but without seedlings) were set up simultaneously and all the treatments and controls were set in triplicate. The exposure reactors used were autoclaved 50 mL brown bottles wrapped with aluminum foil to provide a dark environment to the roots and eliminate photolysis of BDE28. In our preliminary experiments, no clear metabolites were observed at initial exposure concentrations of 5 ng/mL and 10 ng/mL and thus exposure experiments were conducted with initial BDE-28 concentration of 100 ng/mL. Fresh exposure solutions were prepared by adding 40 μL of the BDE-28 working solution to 40 mL autoclaved deionized water (with about 1‰ v/v of methanol in the exposure solution). Then each reactor was planted with three pumpkin seedlings. The mouths of reactors were fitted with aluminum foil and parafilm. All the experiments were conducted in a laminar flow hood. During hydroponic exposure, approximately 1.5 mL/day of autoclaved deionized water was injected into each reactor to supplement transpiration losses. The pumpkins were harvested

Figure 1. Scheme of hydroponic exposure reactor and sampling. A, the leaves; B, the stem out of the reactor; C, the stem in the reactor; and D, the root.

Tissue Culture Exposure. The callus of pumpkin plant were obtained as follows: first, unshelled seeds were ripened in autoclaved deionized water (55 °C) for 1 h, surface sterilized by 75% alcohol for 30 s and by NaClO solution (1% active Cl) for 20 min. Then the seeds were placed on an agarose medium (0.35% in 9 mm culture dish) and cultivated at 30 °C in the dark to grow axenic seedlings. After 5 days, cotyledons were cut into small pieces and placed on solid MS medium (2% sucrose, 2% phytagel, NAA 5 mg/L, 6-BA 3 mg/L, pH 5.6−5.8) in 10 mm culture dishes to induce callus. After 14 days, the callus was collected for the exposure experiment. The inducing of callus and subsequent exposures were all performed at 25 °C in the dark. About 0.5 g of callus were transferred to 3 mL hydroponic MS medium (2% sucrose) which was placed in 5 mL centrifuge tube. The solution and the tubes were all autoclaved before use. The initial exposure concentration of BDE-28 was set at 100 ng/mL. Boiled callus were used as dead controls. Blank controls (without BDE-28) and unplanted controls (without callus) were also set up at the same time. The treated callus and controls were all performed in triplicate. After 96 h, callus and culture solution were sampled to determine the metabolism products. Sample Preparation. All the pumpkin and tissue samples were frozen at −80 °C overnight, freeze-dried, and stored at 13495

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Table 1. Classification of Potential Metabolites group number

metabolites from

I

debromination

II III

bromine rearrangement methoxylation

IV

hydroxyaltion

compounds

formula

number of available standards

number of compounds

mono-BDE di-BDE tri-BDE MeO-mono-BDE MeO-di-BDE MeO-tri-BDE OH-mono-BDE OH-di-BDE OH-tri-BDE

C12H9OBr C12H8OBr2 C12H7OBr3 C13H11O2Br C13H10O2Br2 C13H9O2Br3 C12H9O2Br C12H8O2Br2 C12H7O2Br3

3 12 24 1 2 4 1 2 4

3 12 24 19 64 136 19 64 136

−20 °C until analysis. The water samples were extracted immediately after sampling. Extraction and cleanup procedure for PBDEs, MeO-PBDEs and OH-PBDEs were based on the method described by Wang et al.17 Two nanograms each of BDE-25 and 4′-OH-BDE-17 were added as surrogate standards to the samples prior to extraction. 5′-MeO-BDE-25 (2 ng) and 5′-OH-BDE-25 (2 ng) were added before instrumental analysis as internal standards. These compounds were not found as metabolites in preliminary experiments. The hydroponic solution of in vivo exposure was transferred into 100 mL extraction bottles and the reactors were rinsed with 5 mL hexane/MTBE (1:1, v/v) for 3 times and the organic phase was also transferred to the extraction bottles. After adding another 5 mL of hexane/MTBE, the solution was extracted under manually shaken for 5 min. The organic phase was transferred to clean vials after phase separation. The water sample was re-extracted by another 20 and 10 mL hexane/ MTBE (1:1, v/v). Then the combined extracts were concentrated using a rotary evaporator and redissolved with 1 mL hexane in a 2 mL centrifuge tube. The culture solution of in vitro exposure was extracted by 1 mL hexane/MTBE in the 5 mL centrifuge tube twice and the combined extracts were replaced by 1 mL hexane after drying under gentle flow of nitrogen gas. For plant tissues, about 0.1−0.2 g of sample was filled in a 2 mL centrifuge tube with two steel balls and 0.8 mL hexane/ MTBE (1:1, v/v). Then the tube was subjected to a shocking process under 30 Hz for 1 min in a refiner (QIAGEN, Hilden, Germany). After centrifugation under 5000 r/min for 1 min, the supernatant was transferred into a clean tube. The extraction was repeated twice and the combined extract was dried under a gentle flow of nitrogen gas and redissolved in 1 mL hexane. For all samples, the hexane phase was partitioned with 0.8 and 0.4 mL of NaOH solution (0.5 M in 50% ethanol) twice and the alkaline solution was then transferred into another clean tube. The organic phase contained the neutral analytes including PBDEs and MeO-PBDEs. The alkaline solution which contained OH-PBDEs was acidified with 500 μL of 1 M HCl and extracted twice with 0.4 mL of hexane/MTBE (9:1, v/ v). After the alkaline partition process, the extracts of the water samples were sufficiently clean for direct instrumental analysis. However, the extracts from plant tissue samples needed further cleanup by multilayer column chromatography. The column was filled with 2 g anhydrous Na2SO4 at the top and 3 g Florisil at the bottom, and rinsed by 20 mL of DCM/hexane (1:1, v/v) before use. The neutral components were eluted with 20 mL DCM/hexane (1:1, v/v) and the phenolic components were eluted by DCM/hexane/methanol (50:45:5, v/v). The eluates were dried under a gentle stream of nitrogen, redissolved in 0.1

mL hexane for the neutral components or 0.1 mL acetonitrile for the phenolic components, and then the internal standards were added before instrumental analysis. Instrumental Analysis. An Agilent 6890 gas chromatograph (GC) coupled with a 5973C mass spectrometer (MS) detector (Agilent Technologies, Palo Alto, CA) was used for the analysis of PBDEs and MeO-PBDEs. Samples were injected by a 7683B Series Injector into a DB-5MS column (30 m × 0.25 mm × 0.1 μm J & W Scientific, Folsom, CA) with splitless mode (280 °C). Helium was used as carrier gas at a constant flow of 1.0 mL/min. The oven program started at 90 °C (hold for 2 min), increased by 30 °C/min to 200 °C, then increased at 2 °C/min to 230 °C. The post run was set at 300 °C. Held for 3 min. Quantitative determination by GC−MS (EI) was in the optimized selected ion monitoring (SIM) mode. The parameters are shown in Supporting Information Table S1. Full scan (m/z 250−450) was performed to further confirm the metabolites. Analysis of OH-PBDEs was performed on an Agilent 1290 Series LC system coupled with an Agilent 6460 Triple Quadrupole MS/MS system (ESI) using a C18 column (100 mm × 2.1 mm, particle size 2.2 μm, Thermo Scientific, Waltham, MA). The parameters of performance are the same as that in Sun’s work.12 In brief, the mobile phase consisting of acetonitrile (Solvent A) and water (Solvent B) was in a gradient elution mode with ratio of A:B from 55:45 to 65:35 in 10 min at a flow rate of 0.4 mL/min. The column was equilibrated for 5 min between runs. The column temperature was set at 40 °C and the volume injected onto the column was 10 μL. The optimized MRM parameters are provided in Supporting Information Table S2. The quality assurance included the addition of surrogate standards and injection potential metabolites standards, namely BDE-1, BDE-3, BDE-7, BDE-8, BDE-17, BDE-25, BDE-28, 2′OH-BDE-3, 2′-OH-BDE-7, 3′-OH-BDE-7, 4′-OH-BDE-17, 5′OH-BDE-25, 2′-OH-BDE-28, 3′-OH-BDE-28, 2′-MeO-BDE-3, 2′-MeO-BDE-7, 3′-MeO-BDE-7, 4′-MeO-BDE-17, 5′-MeOBDE-25, 2′-MeO-BDE28 and 3′-MeO-BDE-28, in blank plant samples. The limits of detection (LOD) were in the range of 0.17−3.08 ng/mL for neutral components and 0.054−12.99 ng/mL for phenolic components, respectively. Recoveries of the surrogate standards in plant tissue and solution samples were 65.4% and 56.4% for BDE-25 and 43.3% and 45.2% for 4′OH-BDE-17, respectively. Recoveries of neutral components and phenolic components were within the ranges 70.7−110%, 27.1−35.5% in the matrix spiked samples, respectively. The former recoveries were used to obtain the amounts of compounds with standard while the latter were used for the semiquantification for unknown compounds as discussed in the result and discussion part. 13496

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Figure 2. GC−MS SIM (m/z = 436) chromatograms of (a) root sample of intact exposed pumpkin and (b) exposed callus sample. Full scan MS of (c) MeO-tri-BDE-A, (d) MeO-tri-BDE-B, and (e) MeO-tri-BDE-C.

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RESULTS AND DISCUSSION Analysis of the Potential Metabolites. The potential metabolites of BDE-28 in the exposure systems were classified into four groups, as shown in Table 1, which included debrominated PBDE metabolites,12,14,23−25 tri-BDE metabolites from bromine rearrangement, 14 hydroxylated and methoxylated metabolites12,14,26 with only one hydroxyl or methoxyl group. All the standards of plausible PBDE metabolites (from BDE-1 to BDE-39) are commercially available. However, there are currently only a limited number of available commercial standards for OH- and MeO-PBDEs (Table 1). Therefore, a method which could identify most of these products with the limited standards was established. The available OH- and MeO-BDE standards were used as representative compounds to optimize the instrumental conditions to tentatively determine the different classes of compounds (Table 1). The chromatograms and full scan mass spectra of 39 PBDEs, 7 MeO-PBDEs, and 7 OH-PBDEs were obtained and characterized. For PBDEs and MeO-PBDEs, subsequent identification of different classes of metabolites were mainly based on searching the most abundant isotope molecular ions (as shown in Supporting Information Table S1)

as well as the proportion of the isotope peaks of Br atoms in the molecular structures. For example, if a compound showed certain responses of molecular ion peaks with correct Br isotope ratio (a rough ratio of 1:1 or 1:2:1 for PBDEs and MeO-PBDEs) in the SIM mode but with different retention time from the available standards, it could be identified as belonging to the same class of compounds. Further confirmation of the structure of metabolites was performed in the EI full-scan mode. In addition, the GC chromatograms helped to segment the time windows for different groups of metabolites (retention time range in Supporting Information Table S1). For OH-PBDEs, the molecular ions with a loss of hydrogen atom ([M − H]−) were chosen as the precursor ions for all groups of compounds. The product ions were 79Br and 81 Br for group II, III, IV. For group I, the product ions were chosen as 108 as no clear response of Br in 2′-OH-BDE-3 was seen due to the relative stable structure. The qualitative analysis was performed based on the response of MRM reaction as well as the ratio between 79Br and 81Br which should be close to 1. Semiquantification of the unknown metabolites was performed based on the average selected ion responses of the commercial 13497

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Figure 3. LC−MS/MS MRM chromatograms of standards, exposed pumpkin roots (in vivo samples) and callus samples (in vitro samples).

standards of the same class of compounds as conducted by Haglund et al.5 Neutral Metabolites. The neutral metabolites of BDE-28 include other PBDE and MeO-PBDEs. In this research, BDE12, BDE-15 and BDE-32 were identified in the in vivo and in vitro exposure systems. However, these PBDE congeners were also found in the in vivo and in vitro unplanted controls. The amounts of these BDE congeners were not significantly different between the exposed and the control samples. Further confirmation by injection of the concentrated standard solution into the GC-MS showed that those brominated BDE congeners were present in the commercial BDE-28 standard. It was

deemed that the BDE congeners were impurities of the BDE-28 standard rather than from the plant metabolism of BDE-28. Therefore, no obvious debromination or Br rearrangement processes were observed in both hydroponic intact pumpkin exposure and callus exposure, which was consistent with the findings that PBDE congeners without meta-substituted Br atom (such as BDE-28) could not be debrominated in fish.24 In Wang et al.’s work,14 low concentrations of impurities, including BDE-2, BDE-3, BDE-12, BDE-13, BDE-15, BDE32, and BDE-37, were found in BDE-28 standard. However, some of those congeners were detected in the exposure system at much higher concentrations and thus debromination and Br 13498

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Table 2. Mass Balances and BDE-28 Distribution in the In Vivo (10 days) and In Vitro (96 h) Studies BDE-28 (ng)a reactors

part A

Exposed pumpkin In vivo blank control In vivo unplanted control Exposed Callus In vitro blank control In vitro dead control In vitro unplanted control

1.43 ± 0.17c 1.31 ± 0.46 −d

part B

part C

0.88 ± 0.04 355.2 ± 69.69 0.61 ± 0.16 0.39 ± 0.04 −d −d 144.3 ± 101.2 n.d.e 146.0 ± 42.33 −d

part D

solution

total BDE-28 recoveredb (%)

1400 ± 1376 0.65 ± 0.14 −d

267.8 ± 201.9 1.43 ± 0.88 3020 ± 1190 43.63 ± 9.78 n.d.e 63.92 ± 67.96 162.2 ± 79.14

50.65 −d 75.51 62.64 −d 69.98 54.06

a

Detected mass was recovery corrected. bSummation of different compartment and expressed as the percent of initial BDE-28. The initial mass of BDE-28 was 4000 ng for in vivo study and 300 ng for in vitro study. cMean value ± standard deviation. dNo sample for detection. eNone detectable.

excreted by the roots.12 In in vitro exposure, only one MeOsubstituted metabolite, MeO-BDE-C, was found in the plant callus, probably due to short exposure time (96 h) and relative small biomass of the samples. No MeO-PBDEs were found in all of the in vivo and in vitro controls. These results indicated that MeO-PBDEs were from the biotransformation of BDE-28 by pumpkin plant and callus. Phenolic Metabolites. In this study, coelution of 2′-OHBDE-7 and 3′-OH-BDE-7 is unavoidable and those two compounds had the same MRM mass response (Figure 3) on LC-MS/MS. A peak with the same retention time and mass spectrum as the coeluted peaks of 2′-OH-BDE-7/3′-OH-BDE7 was observed for the in vivo and in vitro exposure samples, suggesting the possible presence of either or both ofthese two compounds. Other OH-substituted metabolites found in both in vivo and in vitro exposure samples showed similar LC−MS/ MS MRM patterns to their corresponding standards but with different retention times. The unknown OH-BDEs were identified as one OH-di-BDE (342.9→78.9, 342.9→80.8) and four OH-tri-BDEs (420.8→78.8, 422.8→80.8, Supporting Information Table S2). Also, the ratios of 79Br and 81Br were close to 1. In the in vivo exposure, hydroxylated metabolites could be found in all parts of the pumpkin and even in the culture solution, although in some samples part A and B did not contain any observable levels of OH-di-BDEs. The total amount of OH-BDEs accounted for about 1.5% (recovery corrected) of initial mass of BDE-28. As shown in Supporting Information Figure S3, the OH-BDE metabolites were mainly distributed in part C and D. The amounts of OH-BDEs in the reactors decreased in the ranking by part D> C> B> solution> A. In in vitro exposure, same hydroxylated metabolites were found in the callus samples (Supporting Information Figure S3). No OH-PBDEs were found in any controls. Therefore, the OH-PBDEs were considered to be formed by the pumpkin rather than from other pathways. The Fate of BDE-28. In exposure samples, 35% of initial mass of BDE-28 was accumulated in part D (roots). About 9% of BDE-28 was enriched in part C, probably by pathways such as translocation from roots and sorption from volatilized proportions in the headspace. About 51% of the initial dose of BDE-28 was recovered at the end of the in vivo exposure (Table 2). The unplanted controls showed a loss of around 25% of initial dose. The loss was likely due to evaporation of BDE-28 because it was found in all the compartments of the in vivo blank controls with the mass distribution pattern of part C < part B ≈ part D < part A ≈ solution. Lower recovery of BDE-28 in the exposed pumpkins than that of unplanted controls

rearrangement processes was considered to have taken place in maize. The interspecific differences between pumpkin and maize may lead to the observed differences on the metabolism of BDE-28 to less brominated congeners. In Sun et al.’s study,12 debromination from BDE-47 (initial exposure concentration of 5 ng/mL) to BDE-28 was found but without further debromination products in young whole pumpkin plant. No clear PBDE metabolites were found at initial exposure with high concentrations of BDE-28 (even at 5 mg/mL) in our preliminary experiments, suggesting that debromination of BDE-28 is more difficult than BDE-47. Three unknown MeO-tri-BDEs (referred as MeO-BDE-A, B, C) were identified. They showed similar responses of the molecular ions for MeO-tri-BDEs and the isotope distributions fit the three Br atoms profile (the ratio of peaks at m/z 436 and 438 was 1:1). Full-scan MS analysis was performed to further study the structure of the detected MeO-tri-BDEs. As shown in Figure 2, clear molecular ions [M]+ and fragment ions [MCH3]+ were observed for all three MeO-BDEs. These two clusters of peaks were used to confirm para-substituted MeOBDEs. Ortho-MeO-PBDEs have typical fragment ions of [MBrCH3]+ and meta-MeO-PBDEs have fragment ions of [M − 2Br]+.7,27 Therefore, the MeO substitutions of the metabolites were at para position relative to the ether bond, consistent with the results of previous studies in which the methoxylated metabolites of BDE-47 in pumpkin and methoxylated metabolites of BDE-209 in fish were also para-MeOPBDEs.12,26 Other work28 suggested that the ortho-MeOPBDEs might have a natural source while meta-MeO-PBDEs and para-MeO-PBDEs may come from the transformation of PBDEs. According to our results, formation of para-MeOPBDEs may be an important process for PBDE metabolism in pumpkin. In intact pumpkin plants, the methoxylated metabolites were detected in the stem in the reactor (part C) and roots (part D), but were not detected in leaves (part A) and the extended stem outside of the reactor (part B). The total MeO-BDEs accounted for about 1.6% (recovery corrected) of initial amount of BDE-28 according to the semiquantitative data. The distribution of MeO-tri-BDEs within the pumpkin plant is shown in Supporting Information Figure S2. MeO-BDE-C mainly accumulated in the roots while MeO-BDE-A mainly accumulated in the stem inside the reactor. The observed limited translocation of MeO-PBDEs from roots to only the stem inside the flask might relate to the properties of the metabolites and the limited exposure time. The origin of the trace amounts of MeO-PBDE metabolites in the hydroponic solution was considered as (i) direct excretion from roots and (ii) role of metabolism of the enzyme and other biomolecules 13499

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indicated that there may have other metabolism processes that have not been accounted for. For in vitro exposure reactors, which were well sealed by caps to minimize the evaporation, loss of BDE-28 still occurred. The tissue samples of dead controls accumulated similar amounts of BDE-28 to that of exposed callus, suggesting that BDE-28 was mainly sorbed to the surface of the callus. Possible Metabolism Pathways. Part A and B did not contain detectable amounts of MeO-PBDEs, and the amounts of BDE-28 were lower than OH-PBDEs based on the semiquantitative data. These results suggested that a majority of hydroxylated metabolites were probably formed in pumpkin roots and then translocated from roots to the shoots. The translocation rate of OH-PBDEs might be faster than that of BDE-28 due to the higher water solubility. Here, three metabolism pathways were hypothesized for the methoxylation and hydroxylation processes: (i) methoxylation and hydroxylation may occur separately at the same time;5 (ii) MeO-PBDEs were formed from methylation of relevant OHPBDEs catalyzed by the cytochrome P450 superfamily;5,29,30 and (iii) OH-PBDEs may be produced from the demethylation of MeO-PBDEs.11,31 OH-tri-BDEs were then further debrominated to form OH-di-BDEs. This was supported by nondetectable amounts of lower brominated products in the exposure reactors. Since all of the methoxylation metabolites were para-MeO-BDEs, a distinctive para-substituted pathway or specific enzyme may be involved in the metabolism of BDE-28 in pumpkin. In conclusion, the hydroxylation and methoxylation of BDE28 under the role of pumpkin were investigated. The methoxylated metabolites of BDE-28 were analogues with MeO-group at para positions relative to the ether bond. No debrominated metabolites were detected. In vitro exposure using pumpkin callus further confirmed that metabolites originated from the plant metabolism rather than from microorganisms. The findings provide very important information on the metabolism of PBDEs, especially lower brominated PBDEs in the environment. In addition, impurities of the standards and the evaporation of the parent compound must be considered when trace metabolites are studied in exposure experiments.

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Molecular structures, instrument parameters, and distribution of metabolites. This material is available free of charge via the Internet at http://pubs.acs.org.

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*Tel: +86 10 62849334. Fax: +86 10 62849339. E-mail: liujy@ rcees.ac.cn. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was jointly supported by the National Basic Research Program of China (nos. 2014CB441105, 2009CB421603), the National Natural Science Foundation of China (no. 21277153), and Important Directional Item of the Chinese Academy of Sciences (no. KZCX2-EW-QN409). 13500

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